MANAGEMENT OF SKIN CANCER BY AGONISTS OF 5-HT1A ANDANTAGONISTS OF - PowerPoint PPT Presentation

MANAGEMENT OF SKIN CANCER BY AGONISTS OF 5-HT1A ANDANTAGONISTS OF 5-HT2A RECEPTORS ANA CATARINA MENEZES, SANDRA SIMÕES, HELENA OLIVEIRA, ANDREIA ASCENSO SUPERVISORS: PROFESSORA DOUTORAANDREIAASCENSO & DOUTORA HELENA OLIVEIRA

PHOTOCARCINOGENESIS Skin cancer is one of the most predominant form of human cancer and its incidence is rapidly increasing around the world in the past few decades Possible causes: Ionizing radiations   Viruses Inflammation  Genetic factors   UV radiation DNA DAMAGE OXIDATIVE STRESS Fig. 1. Exposure to UV radiation and development of melanoma and IMMUNOSUPPRESSION nonmelanoma skin cancers (Menezes AC, et al. Molecular Neurobiology 2015.doi:10.1007/s12035-014-9068-z) 4

PHOTOCARCINOGENESIS The main target of carcinogenesis is the DNA Cyclobutane-type pyrimidine dimers (CPD) 6-4 photoproducts Free radicals Cis -urocanic acid DNA damage Membrane lipid (UCA) peroxidation Immunosuppression Fig. 2. Immunosuppression resulting from exposure to UVB (Menezes AC, et al. Molecular Neurobiology 2015. doi:10.1007/s12035-014-9068-z) 5

MELANOMA  Malignant melanoma is the deadliest form of skin cancer (80% of deaths) and one of the most challenging malignancies to address therapeutically due to its metastatic potential Several mechanisms are involved in chemoresistance of melanoma cells  Counteracting the harmful effects of DNA-damaging drugs Promoting antioxidant adaptive mechanisms DNA photoproducts  Melanoma arises from epidermal melanocytes , which are the main Gene mutations producers of serotonin in the skin and possess both 5-HT1/2A receptors Immunosuppression Oxidative damage Fig. 3. Direct effects of UV exposure (adapted from http://www.skincancer.org/)

SEROTONIN:A KEY MEDIATOR BETWEENTHE SKIN ANDTHE NEUROENDOCRINE SYSTEM T able 1. Serotonergic receptors or serotonin transporter (SERT) Serotonin (5-HT) is biosynthesized from L-tryptophan and is  distribution in human skin of atopic dermatitis patients (Menezes AC, et al. Molecular Neurobiology 2015. doi:10.1007/s12035- widely distributed throughout the body, including the skin 014-9068-z)  Serotonergic membrane-bound receptors are categorized into seven general families from 5-HT1R to 5-HT7R, with at least 21 subtypes 5-HT2AR 5-HT1AR Modulation of immune cells Binding with cis -UCA function 5

THERAPEUTIC POTENTIAL OF SEROTONERGIC DRUGS IN THE PHOTOCARCINOGENESIS CONTEXT  5-HT2AR antagonists are also capable of inducing DNA repair by two different paths Accelerating the repair of both CPD and 6-4 photoproducts Decreasing ROS-induced lesions  5-HT1AR agonists and 5-HT2AR antagonists are capable of preventing UV- and cis -UCA-induced immunosuppression and hence skin cancer induction and progression  1-(1-Naphthyl)piperazine (1-NPZ) is both an agonist of 5-HT1A and antagonist of 5-HT2A receptors with a dual mechanism of action 6

AIMS Assess the potential of a novel therapeutic strategy based on the topical delivery of 1-(1-Naphthyl)piperazine ( 1-NPZ ), both an agonist and antagonist of serotonin receptors (i.e. 5-HT1/2A),towards the treatment of melanoma skin cancer A) Cell Biology Studies B) Pharmaceutical Technology Studies 2

Toxicology and Applied Pharmacology EFFECT OF 1-(1-NAPHTHYL)PIPERAZINE ON HUMAN MNT -1 MELANOMA CELLS ANA CATARINA MENEZES 1 , MANUELA CARVALHEIRO 2 , JOSÉ MIGUEL P . FERREIRA DE OLIVEIRA 3 , ANDREIA ASCENSO 2 , HELENAOLIVEIRA 3 1 Faculdade de Farmácia da Universidade de Lisboa – Lisbon,Portugal 2 NanoBB Research Group of iMed.UL – Lisbon,Portugal 3 Departamento de Biologia,CESAM,Universidade deAveiro – Aveiro,Portugal

METHODS 3/24-h treatment with 24-h treatment with 24-h treatment with 1-NPZ (0-300 µg/mL) 1-NPZ (0,IC 20 ,IC 50 ) 24-h treatment with 1-NPZ (0,IC 20 ,IC 50 ) 1-NPZ (0,IC 50 ) 24-h treatment with Harvested and Harvested, centrifuged 1-NPZ (0,IC 20 ,IC 50 ) Washed and lysed in centrifuged and resuspended in 1x TRIzol reagent binding buffer Incubated for 4h with MTT solution Centrifugations + Chloroform Centrifugations Incubated for 30min Incubated for Nanodrop with DCFH-DA 15min with PI and AnnexinV-FITC Harvested cDNA synthesis Incubated for +1x binding buffer Incubated for 2h 20min with PI with DMSO Flow cytometry and RNase qR T -PCR Absorbance at Flow cytometry MTT assay Flow cytometry 570 nm 9

RESULTS – CELL VIABILITY ASSAY 120 * * ** 100 0 (Control) 141.8 µM (IC 20 ) 163.6 µM (IC 50 ) ** ** Cell Viability(%) 80 3 h 24 h 40 20 ** ** 0 0 50 100 150 200 250 300 Fig. 4. Light microscopy images (100X) of MNT -1 cells exposed to 1-NPZ (0, 141.8 and [1-NPZ] µM 163.6 µM) for 24h Fig. 5. Effects of 1-NPZ exposure on MNT -1 cell viability for 24h. Results are represented as mean ± SD (n = 3). * p <0.05, ** p <0.001  Treatment of human MNT -1 melanoma cells with 1-NPZ caused distinctive morphological changes , such as roundness and flattening 1-NPZ exposure for either 3 or 24h significantly ( p <0.001, p <0.05) inhibited the cell viability in a dose- and time-dependentmanner  10

CELL CYCLE ANALYSIS 70 *  The cellular response to DNA damage is a cell cycle arrest 60 via checkpoint mechanisms, which in turn depend on the type Cell cycle distribution (%) of damage and the phase of the cell cycle at which it occurs Control IC20 30 T reatment of MNT -1 cells with 1-NPZ (IC 50 ) induced a  IC50 significant ( p <0.05) S-phase delay of cell cycle progression 10 At IC 50 , the percentage of cells in sub-G1 phase significantly 0  Sub-G1 G0/G1 S G2/M ( p <0.05) increased, which might be a sign of apoptosis Fig. 6. Effects of 1-NPZ exposure on MNT -1 cell cycle dynamics for 24h. Results are represented as mean ± SD (n = 3). * p <0.05 11

ROS DETECTIONASSAY 35 30 * 25 Control DCF MFI 20 IC20 15 IC50 10 5 0 Fig. 7. DCF mean fluorescence intensity (MFI) after 24-h treatment. Fig. 8. DCF fluorescence intensity following treatment with 1- Results are expressed as mean ± SD (n = 3). * p <0.05 NPZ on MNT-1 cells for 24h Treatment of MNT -1 cells with 1-NPZ significantly ( p <0.05) increased ROS levels in a dose-dependent manner   These results supported the hypothesis that treatment with 1-NPZ would lead to an increase in ROS intracellular levels, which might play an essential role in 1-NPZ-induced apoptosis in cells 12

CELL APOPTOSIS ASSAY 100 IC 20 Control IC 50 90 80 70 ** Cells (%) 60 Control 50 IC20 40 ** IC50 * 30 ** * 20 10 * * 0 Fig. 10. Annexin V-FITC dot-plots gating (FL1 LOG vs FITC LOG) following treatment with 1- NPZ on MNT-1 cells for 24h: Q1 – Necrotic cells (Annexin-FITC (-) and PI (+)); Q2 – Late Viable and non- Early apoptotic Late apoptotic Necrotic apoptotic cells (Annexin-FITC (+) and PI (+)); Q3 – Early apoptotic cells (Annexin-FITC (+) and apoptotic PI (-)); Q4 – Viable and non-apoptotic cells (Annexin-FITC (-) and PI(-)) Fig. 9. Percentage of apoptotic cells after 24 h treatment. Results are expressed as mean ± SD (n = 3). * p <0.05; ** p <0.001 Treating cells with 1-NPZ caused a highly significant ( p <0.001) reduction in viable and non-apoptotic cells as well as a highly significant  ( p <0.001) increase in early apoptotic cells at both concentrations 13

GENE EXPRESSION ASSAY a) b) c) ** Fig. 11. COX-2 (a), IL12A (b) and PAK1 (c) expression after 24-h treatment with 1-NPZ on MNT-1 cells. Results are represented as mean ± SD (n = 3). ** p <0.001 mRNA expression of genes associated with UV-induced immunosuppression ( COX-2 or PTGS2 and IL12A ) increased following  exposure to 1-NPZ, being COX-2 significantly up-regulated( p <0.001) Exposure of MNT -1 cells to 1-NPZ induced a decrease in PAK1 expression, which in turn is related to chemoresistanceevents .  The unexpected increase in COX-2 expression levels might be explained by a similar COX-2-dependent pathway underlying apoptosis.  14

CONCLUSIONS This study showed that 1-NPZ was capable of inhibiting cellular growth by inducing S-phase cell cycle delay, ROS generation and apoptosis in human MNT -1 melanoma cells 1-NPZ treatment was capable of inducing changes on important signaling cascades attending to different expression levels of various genes in exposed cells 15

European Journal of Pharmaceutics and Biopharmaceutics DEVELOPMENT AND CHARACTERIZATION OF NOVEL 1-(1-NAPHTHYL)PIPERAZINE LOADED LIPID VESICLES FOR PREVENTION OF UV-INDUCED SKIN INFLAMMATION ANA CATARINA MENEZES 1 , PATRÍCIA M. CAMPOS 3 , CARLA EULETÉRIO 1 ,FABÍOLA SG PRAÇA 3 , MARIA VITÓRIA LB BENTLEY 3 , SANDRA SIMÕES, 2 ANDREIA ASCENSO 2 1 Faculdade de Farmácia da Universidade de Lisboa – Lisbon,Portugal 2 NanoBB Research Group of iMed.UL – Lisbon,Portugal 3 School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil